The genetic analysis of tumorigenesis is being carried out with the uniquely useful CHEF cell line (Chinese hamster embryo fibroblasts) developed in this laboratory. CHEF/18 is diploid, anchorage-dependent and non-tumorigenic. Carcinogensis studies are continuing with EMS, MNNG, and 4-NQO. Single-step mutants that grow in low serum (LS) or anchorage-independent (anch) will be examined for tumorigenicity, for karyotypic changes, and for acquisition of LS and anch phenotypes by microcell analysis and by DNA transfer (i.e. transfection). All tumors arising in nude mice from chemically treated cells will be excised, characterized, karyotypes analyzed, grown up in mass culture, cloned, and frozen for further study. The numerous LS and anch mutants obtained will be inter-crossed by cell fusion to determine by complementation analysis how many LS genes and anch genes have been detected. With the use of serum-free medium previously described, we will examine the altered growth requirements of LS mutants and of tumor-derived clones. Mapping studies of newly acquired drug-resistant mutants will be continued using microcell analysis, and DNA transfer methods will be applied as they become available.